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BIG PHYSICS, BIG QUESTIONS –

Technology : Gene trap catches `knockout’ mice

By Andy Coghlan

GENETICISTS are to receive a helping hand from 500 000 strains of mutant
mice. Each strain will have a single defective gene, so geneticists can study
the function of the “silenced” gene. By offering strains with defective mouse
equivalents of all 100 000 human genes, a company in Texas hopes to speed up the
search for new drugs and treatments.

“Having this bank of mammalian mutants will greatly accelerate genetic
research,” says Arthur Sands, the president of Lexicon Genetics, the company in
Woodlands, Texas, building the collection.

Mice make good subjects for studying human genetics because the mouse and
human genomes have roughly the same number of genes, the same size of genome and
genes in the same order on the chromosomes. In addition, existing “knockout”
mice show symptoms very similar to those of people who lack the same gene.

There are already some 400 strains of knockout mice. But the work required to
produce them was immense, taking several years. For example, the cancer-prone
p53 knockout mouse, which lacks a gene that normally protects against tumours,
took three years to develop. With the help of new technology, Lexicon hopes to
offer ready-made versions of all possible knockout strains.

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Embryonic cells of each strain will be stored in liquid nitrogen at
–175 °C and grown into mice when needed. Geneticists normally have to
develop each knockout strain from scratch using a complex technique called
homologous recombination.

So far, Lexicon has only produced 200 strains of knockout mice, but it says
its technology will enable it to produce 500 000 strains within five years.

The company intends to supply roughly five strains per gene, some with the
gene function eliminated completely and some with it merely damaged. “Both can
provide useful information to the geneticist,” says Glenn Friedrich, chief
scientist of Lexicon.

An accompanying database will list the DNA sequences corresponding to the
disabled gene in each strain. Researchers studying a particular gene can trawl
the database for similar DNA sequences and order corresponding mouse
strains.

Lexicon is using a new “gene trapping” technology. It will not give full
details of the approach because it has not yet been granted a patent.

The company will say that it relies on specially constructed versions of the
mouse Moloney virus, which normally causes leukaemia in mice. The researchers
altered the virus so that it sabotages mouse genes, but cannot reproduce.

To produce each strain, researchers first harvest primitive cells from
three-day-old mouse embryos, or blastocysts. They then expose these embryonic
stem cells to the engineered virus, which incorporates itself at random into
mouse DNA, sabotaging any gene it hits.

Provided the virus lands in a gene rather than a stretch of “junk DNA”, it
will insert a gene that makes the stem cell resistant to the antibiotic
neomycin. To ensure only cells with disrupted genes survive Friedrich’s team
exposed a population of infected stem cells to neomycin, which killed any other
cells.

Additional genetic machinery incorporated in the virus lets the team recover
fragments of the original, disrupted gene from each culture of mutant stem
cells. By decoding the DNA sequences of these fragments, called expressed
sequence tags, the company can record the specific DNA sequence corresponding to
each mutant strain in its database. The database is essential, says Friedrich,
because it is the only way for researchers examining a particular gene sequence
to trace matching strains.

When a particular strain is ordered, Lexicon will remove the appropriate
cultures from the freezer and inject the cells through a fine needle into a
blastocyst, which is implanted into the womb of an adult mouse. When the mice
are born 20 days later, they each carry one defective copy of the gene.
Researchers who order the mice can then breed full knockouts by mating the
animals they receive. “One-quarter of the litter will have two copies of the
defective gene,” says Friedrich.

Sands says that many strains can be made at once because each individual
virus knocks out a different gene in each stem cell.

“It seems to me like a wonderful concept,” says Klaus Rajewsky, professor of
molecular genetics in the department of biology at the University of Cologne, a
leading group in the development of knockout mice. “It sounds like they will
have all possible knockouts available.”